1. Field of the Invention
This invention is directed to a lyophilized protein formulation, a gel reconstituted from the formulation with water, and a method of topical treatment utilizing the gel.
2. Description of Background and Related Art
In the past ten years, advances in biotechnology have made it possible to produce a variety of proteins using recombinant DNA techniques for pharmaceutical applications. Because of proteolytic degradation in the gastrointestinal tract and poor permeability of these large molecular weight molecules through the intestinal mucosa, oral administration is usually not feasible. Hence, most of these proteinaceous pharmaceutical agents are administered by intravenous, intramuscular, or subcutaneous injections. The parenteral mode of delivery is also desirable for its inherent pulsed administration. Banerjee, P. S., Parenteral Delivery of Peptide and Protein Drugs, in Peptide and Protein Drug Delivery, Advances in Parenteral Sciences: 4. Lee V. H. L., ed. (Marcel Dekker, Inc., New York, N.Y., 1991)
Understandably, this form of administration has been poorly accepted by patients, except for those suffering from life-threatening situations. For this reason, some proteins have recently been evaluated for topical applications, including relaxin for inhibition of uterine myometrial contraction and cervical ripening at parturition, tissue factor for clotting blood, TGF-.beta. for wound healing, and interferon-gamma for atopic dermatitis and trauma-related infections.
Methylcellulose is a synthetic derivative of the naturally occurring cellulose polymer. It differs from cellulose in that two of the three hydroxy groups of the glucose unit are substituted by a methoxy group. This group substitution allows cellulose to hydrate and form a hydrogel. In the pharmaceutical industry, this gel has been commonly used for synthetic drugs and small peptides in topical applications. See, e.g., Howell et al., J. Periodont. Res., 26: 180-183 (1991); Spirtos et al., Gynecol. Oncol., 37: 34-38 (1990); Sacks et al., J. Infect. Dis., 161: 692-698 (1990); Groeneboer et al., Curr. Eye Res., 8: 131-138 (1989); Bohr et al., Arch. Dermatol. Res., 279: 147-150 (1987); Schultz et al., Cornea, 7: 96-101 (1988); Schmidt et al., Acta Ophthalmol., 59: 422-427 (1981).
In addition, cellulose derivatives have been used to formulate therapeutic proteins or polypeptides for topical use. See, e.g., EP 267,015 published May 11, 1988; EP 308,238 published Mar. 22, 1989; and EP 312,208 published Apr. 19, 1989, which disclose formulation of a polypeptide growth factor having mitogenic activity, such as TGF-.beta., in a polysaccharide such as methylcellulose; EP 261,599 published Mar. 30, 1988 disclosing human topical applications containing growth factors such as TGF-.beta.; EP 193,917 published Sep. 10, 1986, which discloses a slow-release composition of a carbohydrate polymer such as a cellulose and a protein such as a growth factor; GB Pat. No. 2,160,528 granted Mar. 9, 1988, describing a formulation of a bioactive protein and a polysaccharide; and EP 193,372 published Sep. 3, 1986, disclosing an intranasally applicable powdery pharmaceutical composition containing an active polypeptide, a quaternary ammonium compound, and a lower alkyl ether of cellulose. See also U.S. Pat. No. 4,609,640 issued Sep. 2, 1986 disclosing a therapeutic agent and a water-soluble chelating agent selected from polysaccharides, celluloses, starches, dextroses, polypeptides, and synthetic polymers able to chelate Ca and Mg; and JP 57/026625 published Feb. 12, 1982 disclosing a preparation of a protein and water-soluble polymer such as soluble cellulose. In addition, a method for entrapping enzymes in gel beads for use as a biocatalyst is described in U.S. Pat. No. 3,859,169. Also, a method for preparing polyvinyl alcohol gel intended as a transdermal vehicle for water-soluble synthetic drugs is disclosed in JP 62/205035 published Sep. 9, 1987.
TGF-.beta. is typically formulated at an acidic pH at which it is active. Various methods for its formulation include adding 2-5% methylcellulose to form a gel [Beck et al., Growth Factors, 3: 267-275 (1990) reporting the effects on wound healing of TGF-.beta. in 3% methylcellulose], adding collagen to form an ointment or suspension [EP 105,014 published Apr. 4, 1984; EP 243,179 published Oct. 28, 1987; EP 213,776 published Mar. 11, 1987], or adding a cosmetically acceptable vehicle to the TGF-.beta. for a topical formulation [U.S. Pat. No. 5,037,643 issued Aug. 6, 1991].
Additionally, proteins other than TGF-.beta. have been formulated with celluloses for various purposes. For example, epidermal growth factor (EGF) is mixed with a water-soluble cellulose polymer to obtain a sterile aqueous medicinal composition. U.S. Pat. No. 4,717,717 issued Jan. 5, 1988. Also, certain pharmaceuticals are mixed with gelatin, lysozyme, albumin or skim milk along with a hydrophilic polymer such as methylcellulose or hydroxypropyl cellulose to improve their absorption or dissolution rate. JP 57/026615 published Feb. 12, 1982. Moreover, corticosteroids can be formulated with gelatin and methylcellulose or other celluloses to form a non-oil ointment. JP 61/233617 published Oct. 17, 1986. In addition, cellulose derivatives have been used as a gel base for relaxin. WO 89/07945 published Sep. 8, 1989. This publication indicates that the formulated relaxin may be in liquid, frozen, or gel form, or may be lyophilized and reconstituted.
It has also been known to mix an active medicament unstable to heat with a biodegradable protein carrier such as collagen, atelocollagen, or gelatin to form a carrier matrix having sustained-release properties. The resultant mixture is then dried, and the dried material is formed into an appropriate shape, as described in U.S. Pat. No. 4,774,091. Examples of active drugs for this purpose are given as t-PA; prostaglandins; prostacyclines; biohormones, e.g., hGH, bGH, GRF, somatomedins, and calcitonin; interferons; interleukins; tumor necrosis factor; and other cytokines such as macrophage activating factor, migration inhibitory factor, and colony stimulating factor.
The major problem with incorporating proteins into gels is the instability of these proteins in such a configuration. For example, when relaxin is incorporated into the liquid methylcellulose gel medium, the protein is stable at 5.degree. C. for only a month even in the absence of light. In addition, for those proteins such as relaxin and TGF-.beta., the liquid containing the protein must be mixed with the gel at the time of administration. This is typically accomplished by the cumbersome and time-consuming procedure of intermixing the two components through syringes connected by an interlocking unit. Insufficient mixing may also lead to therapeutic failure.
For example, the current gel formulation package for relaxin contains one protein vial (lyophilized or liquid), one diluent vial (if the protein is in lyophilized form), one liquid gel vial, two syringes, two needles, and one interlock connector for the syringes. In hospitals, physicians are required to undertake a complicated procedure of mixing the protein and wet gel together, reconstituting the protein vial with diluent, withdrawing reconstituted protein solution into one syringe first, and then withdrawing wet gel into another syringe. After removing needles from both syringes, they connect the syringes with an interlock connector and push two syringe plungers back and forth to allow the gel and protein solution to mix for use.
Freeze-drying, or lyophilization, is a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Rey, "Some basic facts about freeze drying," p. xiii, in Goldblith et al., ed., Freeze Drying and Advanced Food Technology, Academic Press, London, 1975; Pikal, Freeze-drying of Proteins, parts 1 and 2, BioPharm, Sep. 1990, p. 18-27 and Oct. 1990, p. 26-30. An excipient may be added to a formulation to be freeze dried so as to reduce the time for reconstitution. Examples of such agents include a sugar, polyol, amino acid, methylamine, or lyotrophic salt. See, e.g., Carpenter and Crowe, "The Mechanism of Cryoprotection of Proteins by Solutes," Cryobiology, 25: 244-255 (1988).
Various small molecular weight drugs have been formulated and then freeze-dried. A dried composition and method of oral administration of drugs, biologicals, nutrients, and foodstuffs is described in U.S. Pat. 5,039,540; however, while the term freeze drying is used, the patent employs a different process entirely. U.S. Pat. No. 4,883,785 discloses a method for preparing a formulation for anti-fungal agent such as amphotericin B, where cyclodextrin is used to improve the solubility of the drug, and lyophilization is employed to preserve the formulation in the solid state.
A freeze-drying method for preparing dry sponge-like polymeric carriers is described in JP 56/25211 published Jun. 24, 1980 by which embedded enzymes (lysozyme, dextranase, mutanase, levanase) can be slowly released when applied to the affected area of the mucous membrane inside the mouth in treating oral cavity and naval cavity diseases.
Moreover, a method is disclosed for preparing lyophilized cellulose lamellae containing low molecular weight molecules, such as pilocarpine chloride, for slow release in the ophthalmic field. Zaloukal, Ceskoslovenska Farmacie, 39: 433-435 (1990).
In contrast to small molecular weight drugs, proteins have a high molecular weight associated with extensive secondary, tertiary, and quaternary structure that can be disrupted upon lyophilization. Hence, proteins tend to aggregate and/or deamidate when treated under certain conditions. There is a special need in the art for a topical, water-soluble polysaccharide-based gel formulation of protein that is stable, does not require complicated mixing procedures before application, and rehydrates readily from a powder reconstituted in water.
Accordingly, it is an object of the present invention to provide a topical gel formulation for proteins that is stable for shipping and storage and is easy to prepare prior to administration.
It is another object to provide a lyophilized formulation of protein that does not require a long time for reconstitution.
It is a further object to provide a formulation package having only one product vial (containing lyophilized protein and gel base), one diluent vial, one syringe, and one needle. Such a package provides advantages in product packaging and manufacturing costs.
These and other objects will become apparent to one of ordinary skill in the pharmaceutical, pharmacologic, veterinary, and clinical arts.